Coordinated ATP hydrolysis by the Hsp90 dimer.

The Hsp90 dimer is a molecular chaperone with an unusual N-terminal ATP binding site. The structure of the ATP binding site makes it a member of a new class of ATP-hydrolyzing enzymes, known as the GHKL family. While for some of the family members structural data on conformational changes occurring after ATP binding are available, these are still lacking for Hsp90. Here we set out to investigate the correlation between dimerization and ATP hydrolysis by Hsp90. The dimerization constant of wild type (WT) Hsp90 was determined to be 60 nm. Heterodimers of WT Hsp90 with fragments lacking the ATP binding domain form readily and exhibit dimerization constants similar to full-length Hsp90. However, the ATPase activity of these heterodimers was significantly lower than that of the wild type protein, indicating cooperative interactions in the N-terminal part of the protein that lead to the activation of the ATPase activity. To further address the contribution of the N-terminal domains to the ATPase activity, we used an Hsp90 point mutant that is unable to bind ATP. Since heterodimers between the WT protein and this mutant showed WT ATPase activity, this mutant, although unable to bind ATP, still has the ability to stimulate the activity in its WT partner domain. Thus, contact formation between the N-terminal domains might not depend on ATP bound to both domains. Together, these results suggest a mechanism for coupling the hydrolysis of ATP to the opening-closing movement of the Hsp90 molecular chaperone.     

Heat shock protein 27 as a prognostic and predictive biomarker in pancreatic ductal adenocarcinoma

A role of heat shock protein 27 (HSP27) as a potential biomarker has been reported in various tumour entities, but comprehensive studies in pancreatic cancer are lacking. Applying tissue microarray (TMA) analysis, we correlated HSP27 protein expression status with clinicopathologic parameters in pancreatic ductal adenocarcinoma specimens from 86 patients. Complementary, we established HSP27 overexpression and RNA-interference models to assess the impact of HSP27 on chemo- and radiosensitivity directly in pancreatic cancer cells. In the TMA study, HSP27 expression was found in 49% of tumour samples. Applying univariate analyses, a significant correlation was found between HSP27 expression and survival. In the multivariate Cox-regression model, HSP27 expression emerged as an independent prognostic factor. HSP27 expression also correlated inversely with nuclear p53 accumulation, indicating either protein interactions between HSP27 and p53 or TP53 mutation-dependent HSP27-regulation in pancreatic cancer. In the sensitivity studies, HSP27 overexpression rendered HSP27 low-expressing PL5 pancreatic cancer cells more susceptible towards treatment with gemcitabine. Vice versa, HSP27 protein depletion in HSP27 high-expressing AsPC-1 cells caused increased gemcitabine resistance. Importantly, HSP27 expression was inducible in pancreatic cancer cell lines as well as primary cells. Taken together, our study suggests a role for HSP27 as a prognostic and predictive marker in pancreatic cancer. Assessment of HSP27 expression could thus facilitate the identification of specific patient subpopulations that might benefit from individualized treatment options. Additional studies need to clarify whether modulation of HSP27 expression could represent an attractive concept to support the incorporation of hyperthermia in clinical treatment protocols for pancreatic cancer.     

FK506-binding protein 52 phosphorylation: a potential mechanism for regulating steroid hormone receptor activity

Functional maturation of steroid hormone receptors requires ordered assembly into a large multichaperone complex consisting of receptor monomer, an Hsp90 dimer, the p23 cochaperone, and an FK506-binding protein (FKBP) family member or alternate peptidylprolyl isomerase-related cochaperone. Previous cellular studies demonstrated that FKBP52 can potentiate receptor function. These results have been confirmed in fkbp4 gene knockout mice in which males are partially androgen insensitive and females display characteristics of progesterone insensitivity. Conversely, FKBP51, which has a high degree of similarity to FKBP52, antagonizes FKBP52-mediated potentiation. Both proteins consist of three domains: two FKBP12-like domains termed FK1 and FK2 and a tetratricopeptide repeat domain that targets binding to Hsp90. To help understand why the two FKBPs behave differently and to gain insight into FKBP52 potentiation activity, we have analyzed the loop structure that links FK1 and FK2. Within the FK linker of FKBP52 is the sequence TEEED, which forms a consensus casein kinase II phosphorylation site; the corresponding sequence in FKBP51 is FED. We demonstrate that the distinct FK linker sequences per se do not account for lack of potentiation activity by FKBP51. However, phosphorylation of the FK linker appears to be an important regulatory determinant of FKBP52-mediated potentiation of steroid receptor activity.     

Successful strategy for the selection of new strawberry-associated rhizobacteria antagonistic to Verticillium wilt

In order to isolate and characterize new strawberry-associated bacteria antagonistic to the soil-borne pathogenic fungus Verticillium dahliae Kleb., rhizobacterial populations from two different strawberry species, Greenish Strawberry (Fragaria viridis) and Garden Strawberry (F. x ananassa) obtained after plating onto King's B and glycerol-arginine agar, were screened for in vitro antagonism toward V. dahliae. The proportion of isolates with antifungal activity determined in in vitro assay against V. dahliae was higher for the Garden Strawberry than for the Greenish Strawberry. From 300 isolates, 20 isolates with strong antifungal activity were selected characterized by physiological profiling and molecular fingerprinting methods. Diversity among the isolates was characterized with molecular fingerprints using amplified ribosomal DNA restriction analysis (ARDRA) and the more discriminating BOX-PCR fingerprint method. The physiological profiles were well correlated with molecular fingerprinting pattern analysis. Significant reduction of Verticillium wilt by bacterial dipping bath treatment was shown in the greenhouse and in fields naturally infested by V. dahliae. The relative increase of yield ranged from 117% (Streptomyces albidoflavus S1) to 344% (Pseudomonas fluorescens P10) in greenhouse trials, and 113% (Streptomyces albidoflavus S1) to 247% (Pseudomonas fluorescens P6) in field trials. Evaluation resulted in the selection of three effective biocontrol agents (Pseudomonas fluorescens P6, P10, and Streptomyces diastatochromogenes S9) antagonistic to the Verticillium wilt pathogen.     

Identification of the phosphorylation sites of the murine small heat shock protein hsp25.

Native phosphorylated mouse small heat shock protein hsp25 from Ehrlich ascites tumor cells was isolated and the in vivo phosphorylation sites of the protein were determined. Furthermore, native hsp25 was phosphorylated by the endogenous kinase(s) in a cell-free system as well as recombinant hsp25 was phosphorylated in vitro by protein kinase C and catalytic subunit of cAMP-dependent protein kinase. The two major phosphorylation sites of native and recombinant hsp25 were determined as Ser-15 and Ser-86. There are no differences in the hsp25 phosphorylation sites phosphorylated by the protein kinase C, the catalytic subunit of cAMP-dependent protein kinase and the unknown intracellular kinase(s). The serine residues identified exist in all known small mammalian stress proteins and are located in the conserved kinase recognition sequence Arg-X-X-Ser.     

Ganglioside-induced CD4 endocytosis occurs independent of serine phosphorylation and is accompanied by dissociation of P56lck.

Gangliosides induce a selective and complete modulation of CD4 from the surface of T cells. CD4 down-modulation occurs by CD4 endocytosis. This process is independent of serine phosphorylation of the cytoplasmic tail of CD4 and does not require the association between the tyrosine protein kinase p56lck and the cytoplasmic tail of CD4. Ganglioside-induced CD4 endocytosis is accompanied by the loss of p56lck activity associated with CD4. Sequential immunoprecipitation analysis using an anti-CD4 antibody and an anti-p56lck antiserum showed that this is caused by the dissociation of the enzyme from the cytoplasmic tail of CD4. The kinetics of p56lck dissociation after ganglioside treatment is identical to that of CD4 endocytosis, suggesting that p56lck is displaced in the process of endosome formation. The results indicate that CD4 endocytosis alone can cause the dissociation of the p56lck complex without the requirement for CD4 phosphorylation.     

Expression of cytokine mRNA and protein in joints and lymphoid organs during the course of rat antigen-induced arthritis

Cytokine expression was assessed during antigen-induced arthritis (AIA) in synovial membrane (SM), inguinal lymph node (LN), and spleen using competitive RT-PCR and sandwich ELISA. In the SM, early elevations of IL-1β and IL-6 mRNA (by 6 hours; 450- and 200-fold, respectively) correlated with the joint swelling; a 6-fold increase in tumor necrosis factor α (TNFα) was not significant. Not only IL-2 and IFN-γ (which increased 10,000-fold and 200-fold, respectively), but also IL-5 and IL-10, increased acutely (6 hours – day 1; 3-fold and 35-fold, respectively) in the SM. In general, the protein levels in the SM for IL-1β, IL-6, TNFα, IFN-γ, IL-4, and IL-10 (increase from 4-fold to 15-fold) matched the course of mRNA expression. In the inguinal LN, there were early mRNA elevations of IL-6 (a 2.5-fold increase by 6 hours, which correlated positively with the joint swelling) and IL-2 (4-fold by 6 hours), as well as later rises of IL-4 and IL-5 (2.5- and 4-fold, respectively, by day 3). No significant elevations of the corresponding proteins in this tissue were observed, except for IL-1β (by day 6) and IL-10 (by day 1). In the spleen, there were significant mRNA elevations at 6 hours of IL-1β (1.5-fold), IL-6 (4-fold; positively correlated with the joint swelling), IFN-γ (3-fold), and IL-2 (7- to 10-fold). IL-5 and IL-10 (2- and 3-fold, respectively) peaked from 6 hours to day 3 in the spleen. Increases of the corresponding proteins were significant in comparison with day 0 only in the case of IL-2 (day 6). By day 6 (transition to the chronic phase), the mRNA for cytokines declined to or below prearthritis levels in all the tissues studied except for IL-1β in the SM and IL-6 in the spleen. AIA is thus characterized by four phenomena: early synovial activation of macrophages, T helper (Th)1-like, and Th2-like cells; late, well-segregated Th2-like responses in the inguinal LN; late, overlapping Th1-like/Th2-like peaks in the spleen; and chronic elevation of synovial IL-1β mRNA and spleen IL-6 mRNA.     

Hsp90 regulates the activity of wild type p53 under physiological and elevated temperatures

The activity and structural integrity of the tumor suppressor protein p53 is of crucial importance for the prevention of cancer. p53 is a conformational flexible and labile protein, in which structured and unstructured regions function in a synergistic manner. The molecular chaperone Hsp90 is known to bind to mutant and wild type p53 in vivo. Using highly purified proteins we analyzed the interaction and the binding sites between both proteins in detail. Our results demonstrate that Hsp90 binds to a folded, native-like conformation of p53 in vitro with micromolar affinity. Specifically, the DNA-binding domain of p53 and the middle and carboxy-terminal domains of Hsp90 are responsible for this interaction, which is essential to stabilize p53 at physiological temperatures and to prevent it from irreversible thermal inactivation. Our results are in agreement with a model in which Hsp90 is required to maintain the folded, active state of p53 by a reversible interaction, thus introducing an additional level of regulation.     

A domain in the N-terminal part of Hsp26 is essential for chaperone function and oligomerization

Small heat-shock proteins (Hsps) are ubiquitous molecular chaperones which prevent the unspecific aggregation of non-native proteins. For Hsp26, a cytosolic sHsp from of Saccharomyces cerevisiae, it has been shown that, at elevated temperatures, the 24 subunit complex dissociates into dimers. This dissociation is required for the efficient interaction with non-native proteins. Deletion analysis of the protein showed that the N-terminal half of Hsp26 (amino acid residues 1-95) is required for the assembly of the oligomer. Limited proteolysis in combination with mass spectrometry suggested that this region can be divided in two parts, an N-terminal segment including amino acid residues 1-30 and a second part ranging from residues 31-95. To analyze the structure and function of the N-terminal part of Hsp26 we created a deletion mutant lacking amino acid residues 1-30. We show that the oligomeric state and the structure, as determined by size exclusion chromatography and electron microscopy, corresponds to that of the Hsp26 wild-type protein. Furthermore, this truncated version of Hsp26 is active as a chaperone. However, in contrast to full length Hsp26, the truncated version dissociates at lower temperatures and complexes with non-native proteins are less stable than those found with wild-type Hsp26. Our results suggest that the N-terminal segment of Hsp26 is involved in both, oligomerization and chaperone function and that the second part of the N-terminal region (amino acid residues 31-95) is essential for both functions.